Discovery and Development of Broad-spectrum Protease Inhibitors of Flaviviruses of Significant Public Health Threats

具有重大公共卫生威胁的黄病毒广谱蛋白酶抑制剂的发现和开发

• 批准号: 9907267
• 负责人: Sridhar G Prasad
• 金额: $ 30万
• 依托单位: PLEX PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907267
• 关键词: Adult; Affect; American; Animal Model; Animals; Antiviral Agents; Arbovirus Infections; Arboviruses; Arthrogryposis; Aspartic Endopeptidases; Binding; Biochemical; Biological Assay; Biological Availability; Bite; Categories; Cell Line; Cells; Cerebral Calcification; Cessation of life; Child; Clinical; Country; Culicidae; Dengue Vaccine; Dengue Virus; Development; Disease; Disease Outbreaks; Drug Kinetics; Encephalitis; Enzymes; Epidemic; Etiology; Evaluation; Evolution; Exhibits; Family; Family member; Fever; Flaviviridae; Flavivirus; Frequencies; Genome; Geography; Guillain-Barré Syndrome; HIV-1; Hepatitis C virus; Human; In Vitro; Incidence; Infant; Infection; Infection prevention; Japanese encephalitis virus; Lead; Libraries; Ligands; Link; Liver; Luciferases; Measures; Meningitis; Metabolism; Microcephaly; Microsomes; Modeling; Mosquito-borne infectious disease; National Institute of Allergy and Infectious Disease; National Security; Neurons; Nonstructural Protein; Paralysed; Pathology; Patients; Peptide Hydrolases; Performance; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Plaque Assay; Polyproteins; Population; Pregnant Women; Prevention; Preventive Intervention; Process; Property; Protease Inhibitor; Public Health; Rattus; Replicon; Reporter; Risk; Safety; Series; Serine Protease; Serotyping; Severities; Solubility; Structure; System; Testing; Therapeutic; Therapeutic Index; Therapeutic Intervention; Thrombocytopenia; Translating; Vaccines; Vector-transmitted infectious disease; Vero Cells; Viral; Viral Encephalitis; Virus; West Nile viral infection; West Nile virus; World Health Organization; Yellow fever virus; ZIKV infection; Zika Virus; abortion; absorption; acute toxicity; analog; anti-viral efficacy; base; calcification; channel blockers; chemotherapeutic agent; congenital zika syndrome; cytotoxicity; design; effective therapy; efficacy study; efficacy trial; global health; in vitro activity; in vivo; inhibitor/antagonist; innovation; lead optimization; member; mortality; mosquito-borne; novel; pathogen; screening; small molecule; small molecule inhibitor; stillbirth; success; targeted agent; three dimensional structure; vaccine candidate

Project Summary Mosquito-borne members of the Flavivirus family including Zika virus (ZKV), Dengue virus (DNV) and West Nile virus (WNV), are classified as re-emerging pathogens due to the frequency and severity of recent epidemics. Also known as arboviruses, these viruses are the etiologic agents of many debilitating diseases affecting the human population worldwide. Consequently, vector borne diseases now account for 17% of all infections worldwide. DNV is the fastest growing arboviral disease currently affecting 400 million annually with 96 million cases manifesting into clinical severity and 22,000 deaths, mainly children. WNV is considered the most important causative agent of viral encephalitis worldwide. The recent ZKV infection outbreak has been associated with congenital microcephaly and intracranial calcification and, in adults, with GBS and severe thrombocytopenia. Currently there is no effective treatment for infections caused by these viruses, which highlights the urgent need to find preventive and therapeutic interventions. The Flaviviridae genome is translated into a single polyprotein which is processed to yield 3 structural and 7 nonstructural proteins. The correct processing of the polyprotein is essential for replication of all flaviviruses, which requires both host proteases and the highly conserved viral NS2B-NS3 protease (NS2B-NS3pro). Hence, the viral protease is a rational target for development of small molecule inhibitors that block flavivirus replication. Small molecule antivirals targeting HIV-1-encoded and HCV-encoded proteases have been successfully developed, which supports the concept of developing chemotherapeutic agents targeting the flavivirus NS2B-NS3pro. The innovation of our proposal is: (i) optimization of a highly-sensitive screening assay for the identification of low binding fragment hits; (ii) evolution of fragment and compound hits into broad-spectrum leads; (iii) the use of replicon and plaque assays to test for cellular efficacy and guide optimization. Our preliminary results and the use of multiple cell-based models supports the feasibility of the discovery of broad-spectrum anti-flaviviral therapeutics. The specific aims are: Aim 1: Complete the screening of fragment and compound libraries for the identification of broad-spectrum NS2B-NS3pro hits. Milestone 1: Identify 6-8 structurally distinct broad-spectrum NS2B-NS3pro hits with an IC50 ≤ 25μM. Aim 2a: Iterative 3D-structure and SAR-based discovery of three non-overlapping broad-spectrum NS2B-NS3pro inhibitor series, using a combination of: (i) commercial analogues and (ii) med-chem design and synthesis approach. Milestone 2: Identify three non-overlapping broad-spectrum NS2B- NS3pro inhibitor series with an IC50 ≤ 200nM. Aim 2b: Characterize biochemically potent inhibitors for: (a) mode of inhibition and (b) enzyme:ligand interactions and prioritize compounds with IC50 ≤ 200nM, for cellular efficacy studies. Aim 3a: In vitro evaluation and optimization of biochemically potent compounds for cellular efficacy (EC50) and cytotoxicity (CC50). Milestone 3: Identify 4-6 lead compounds from each series with EC50 ≤ 5μM and CC50 ≥ 200μM. Aim 3b: Conduct in vitro ADME-based lead optimization of compounds with broad spectrum in vitro activity and acceptable therapeutic indices. Phase I Milestone: Identify 2-4 lead compounds exhibiting high bioavailability, weak inhibitor of CYPP450s, optimum stability and are not hERG channel blockers.

项目摘要 蚊子传播的黄病毒家族成员，包括寨卡病毒（ZKV），登革热病毒（DNV）和西尼罗河病毒 由于最近流行病的频率和严重性，西尼罗河病毒（WNV）被归类为重新出现的病原体。还已知 作为虫媒病毒，这些病毒是影响人类的许多致衰弱疾病的病原体 国际吧因此，媒介传播疾病现在占全世界所有感染的17%。DNV最快 日益增长的虫媒病毒疾病目前每年影响4亿人，其中9600万例表现为临床严重程度 22,000人死亡，主要是儿童。西尼罗河病毒被认为是世界范围内病毒性脑炎最重要的病原体。 最近的ZKV感染爆发与先天性小头畸形和颅内钙化有关， 成人，GBS和严重血小板减少症。目前还没有有效的治疗方法来治疗由这些引起的感染。 这突出表明迫切需要找到预防和治疗干预措施。 黄病毒科基因组被翻译成单个多蛋白，其被加工以产生3个结构蛋白和7个结构蛋白。 非结构蛋白多蛋白的正确加工对于所有黄病毒的复制是必不可少的，这需要 宿主蛋白酶和高度保守的病毒NS 2B-NS 3蛋白酶（NS 2B-NS 3 pro）。因此，病毒蛋白酶是一种 开发阻断黄病毒复制的小分子抑制剂的合理靶点。小分子抗病毒药物 已经成功开发了靶向HIV-1编码和HCV编码的蛋白酶，这支持了 开发靶向黄病毒NS 2B-NS 3 pro的化疗剂。我们的建议的创新之处在于：（i） 优化用于鉴定低结合片段命中的高灵敏度筛选测定;（ii） 片段和化合物命中广谱线索;（iii）使用复制子和噬斑测定来测试细胞功效 引导优化。我们的初步结果和使用多个基于细胞的模型支持的可行性， 发现广谱抗黄病毒疗法。具体目标为：目标1：完成片段的筛选 和用于鉴定广谱NS 2B-NS 3 pro命中的化合物文库。里程碑1：从结构上确定6-8 不同的广谱NS 2B-NS 3 pro命中，IC 50 ≤ 25μM。目标2a：迭代3D结构和基于SAR的发现 三个非重叠的广谱NS 2B-NS 3 pro抑制剂系列，使用以下的组合：（i）商业类似物 和（ii）med-chem设计和合成方法。里程碑2：确定三个不重叠的广谱NS 2B- NS 3 pro抑制剂系列，IC 50 ≤ 200 nM。目的2b：表征生物化学上有效的抑制剂：（a） 抑制和（B）酶：配体相互作用，并优先选择IC 50 ≤ 200 nM化合物，用于细胞功效研究。 目的3a：用于细胞功效（EC 50）的生物化学有效化合物的体外评价和优化， 细胞毒性（CC 50）。里程碑3：从每个系列中鉴别出4-6种EC 50 ≤ 5μM和CC 50 ≥ 200μM的先导化合物。目的 3b：对具有广谱体外活性且可接受的化合物进行基于ADME的体外先导优化 治疗指标I期里程碑：确定2-4种具有高生物利用度、弱抑制剂的先导化合物。 CYPP 450具有最佳稳定性，不是hERG通道阻滞剂。